Macrophages play important roles in host homeostasis and pathogenesis of diseases, such as clearance of cellular debris and pathogens [1], metabolic function [2], and immune response [3]. Kupffer cells (KCs) are predominant macrophages in healthy liver, which can self-renew or proliferate [4]. KCs reside along with liver sinusoidal endothelial cells (LSECs) and protect liver infection and injury with functions of phagocytosis, regulation of immunological tolerance, and metabolic function [5]. However, in pathogenic conditions, monocyte-derived macrophages from bone marrow can be recruited into the liver and be differentiated into macrophages and KC-like macrophages [6]. For example, In the development of metabolic dysfunction-associated fatty liver disease (MAFLD), liver resident KCs are lost and replaced by C-type lectin domain family 4 member F negative (CLEC4F⁻) monocyte-derived macrophages expressing osteopontin [7]. Monocyte-derived macrophages express high levels of lymphocyte antigen 6 complex (Ly6c) and C-C motif chemokine receptor 2 (CCR2). In addition, there is a very small population of macrophages different from KCs and monocytes-derived macrophages, including capsular macrophages and peritoneal macrophages in the liver [8].

Current studies using the single-cell RNA-sequencing (scRNA-seq) technique have revealed the presence of multiple macrophage subsets with distinct functions in various tissues [9]. Recently, Blériot et al. [10] reported that there are two phenotypically and functionally distinct populations of liver resident KCs in healthy mice, a major cluster of differentiation 206 low expression (CD206^low)endothelial cell-selective adhesion molecule negative (ESAM⁻) population (KC1) and a minor CD206^highESAM⁺ population (KC2). Both populations express liver resident macrophage markers such as adhesion G protein-coupled receptor E1 (Adgre1) encoding F4/80, integrin subunit alpha M (ITGAM) encoding CD11b, T-cell membrane protein 4 (Tim4), Clec4f, and colony-stimulating factor 1 receptor (Csf1r) (Figure 1). In contrast, KC2 expressed markers like LSECs, such as CD206, CD31 [platelet endothelial cell adhesion molecule 1 (Pecam1, also known as CD31)], lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1), and ESAM. The frequency of KC2 was increased in the livers of mice fed a high-fat diet compared to that in mice fed a normal diet. The authors also found that functional depletion of KC2 or silencing fatty acid transporter CD36 resulting in a reduction of both reactive oxygen species (ROS) and the lipid peroxidation byproduct malondialdehyde (MDA), showing that KC2 functions on hepatic lipid peroxidation and oxidative stress [10].

ESAM belongs to the immunoglobulin superfamily, which is expressed in vascular endothelial cells and plays pivotal roles in angiogenesis and cell migration [11]. ESAM is also expressed in dendritic cells (DCs), which is associated with the expression of another immunoglobulin superfamily protein signal regulatory protein α (SIRPα) [12]. In addition, ESAM expression in hematopoietic progenitors plays a critical role in the erythroid recovery after a bone marrow injury [13].

CD206, the macrophage mannose receptor, is highly expressed in the alternatively activated macrophages (anti-inflammatory phenotype) [9]. In addition, CD206 is also expressed by DCs and LSECs in the liver, playing an essential role in endocytosis and phagocytosis [14]. Modulating a conformational change of CD206 by a synthetic amphipathic peptide sequence: KFRKAFKRFF (RP-182) can drive alternatively activated macrophages (M2-like) tumor-associated macrophages (TAMs) to an antitumor classically activated macrophages (M1-like) phenotype to enhance innate anti-tumor immunity [15]. However, the accumulation of CD206⁺ macrophages expressing tumor necrosis factor-α (TNF-α) and granulocyte-macrophage colony-stimulating factor (GM-CSF) was shown in human fibrotic liver, and these macrophages were differentiated from monocytes stimulated by GM-CSF [16]. In vitro study further illustrated that lipopolysaccharide (LPS) induced monocytes conversion to CD206⁺ macrophage-like cells, which caused an increase in production of TNF-α that contributes to chronic inflammation [16].

Macrophage is one of the antigen-presentation cells, including KCs [17]. Collaborating with Blériot and co-workers, De Simone et al. [18] also found that CD45⁺F4/80⁺CD11^bintTIM-4⁺KCs consist of CD206⁻ESAM⁻KC1 (about 70–85%) and CD206⁺ESAM⁺KC2 (about 15–30%), another commentary paper. Specifically, KC2 can cross-present hepatocellular antigens to prime antiviral function of CD8⁺ T cells by sensing interleukin-2 (IL-2) in hepatitis B virus (HBV) replication-competent transgenic mice [18]. In addition, depletion of KC2 abrogated the function of IL-2 to revert the dysfunction of T cells.

Macrophages play pivotal roles in liver homeostasis in physiological conditions and chronic liver diseases, including non-alcoholic fatty liver disease (NAFLD), MAFLD, liver fibrosis, and end-stage hepatocellular carcinoma (HCC) [19, 20]. The major and significant difference between MAFLD and NAFLD is that NAFLD excludes some metabolic abnormalities with other etiologies that cause liver diseases such as alcohol-associated and viral-associated liver diseases, while MAFLD does not [21, 22]. For example, inhibiting the infiltration of CD206⁺ macrophages in the liver treated with an anti-GM-CSF neutralizing antibody can ameliorate liver fibrosis in HBV-infected humanized mice [16]. In addition, inhibiting the accumulation of monocyte-derived macrophages [e.g., blocking C-C motif chemokine ligand 2 (CCL2)/CCR2] signaling pathway can prevent liver inflammation, NAFLD, and liver fibrosis [23, 24]. Furthermore, macrophage markers such as soluble CD163 (sCD163) that is associated with liver inflammation and glucose metabolism in NAFLD, can be applied to assist diagnosis [25].

In summary, differentiating specific populations of hepatic macrophages is critically important for NAFLD and/or MAFLD treatments and their distinct expressing markers may be applied for liver disease diagnosis.